Effects And Mechanisms Of Combination Of The Afatinib And Enzalutamide In Prostate Cancer Progression

Background:Prostate cancer is the second commonest malignant tumor in males worldwide,which can cause deaths more than 385560 globally.Enzalutamide,the FDA approved second generation androgen receptor anagonist,is widely used in metastatic castration refractory prostate cancer or non-metastatic castration refractory prostate cancer,which succeeds in prolonging ths overall survival of patients generally.However,with the developments of enzalutamide,drug resistance also appeared.Objectives:To verify the combination effect of the tyrosine kinase inhibitor afatinib and enzalutamide on the biological function and mechanism of prostate cancer in vitro.Methods:1.Cbioportal,ualcan and gepia were used to analyze the expression differences,mutation rate and clinical correlation between tyrosine kinase and prostate cancer in TCGA/MSKCC databases.2.The MTT assay were used to evaluate the combination effect on proliferation of different tyrosine kinase inhibitors(gefitinib,erlotinib,sorafenib,afatinib and dacomitinib)and enzalutamide on human prostate cancer cell lines.3.Flow cytometry were used to evaluate the combination effect of the tyrosine kinase inhibitor afatinib and enzalutamide on cell cycle,apoptosis ability.4.Transwell assay was used to evaluate the combination effect of the tyrosine kinase inhibitor afatinib and enzalutamide on invasion capability of tumor cells.5.Western blot assay were used to detect the effect of the tyrosine kinase inhibitor afatinib combined with enzalutamide on the expression of tyrosine kinase,androgen receptor,cell apoptosis,cell cycle and EMT pathway proteins.Result:1.Among a variety of tyrosine kinase inhibitors,afatinib had the best anti-tumor activity,with IC50 reaching the level of nanomole.2.The combination of the tyrosine kinase inhibitor afatinib and enzalutamide not only inhibited the proliferation of prostate cancer cells,but also repressed the invasion ability of tumor cells.This combination also blocked the cycle of prostate cancer cells in G2/M phase and induced cell apoptosis.3.Western blot assay further showed that after treatment with enzalutamide on prostate cancer cell,the protein expression of AR and PSA downregulated,but the protein expression of HER2 and the protein phosphorylation level of Akt increased.However,with treatment on prostate tumor cell with afatinib,it could significantly inhibited the protein expression of HER2 and HER3,also downregulated the protein phosphorylation level of EGFR and Akt.On the other hand,the combination of the tyrosine kinase inhibitor afatinib and enzalutamide could significantly downregulate the protein expression of Bcl-2 and upregulate the protein expression of Bax.Meanwhile,it also showed some repression effects on the protein expression of P53 and P21.What’s more,the combination of the tyrosine kinase inhibitor afatinib and enzalutamide showed some repression effects on migration capacity of prostate cancer cells,which reflected the upregulation of protein expression of E-cadherin and downregulation of protein expression of N-cadherin.Conclusions:1.In this study,the drug afatinib with significant inhibitory effect on prostate cancer tumor cells were screen out from tyrosine kinase inhibitors.And the combined application of afatinib and enzalutamide had significant synergistic inhibitory effect on the biological function of prostate cancer cell proliferation and cell invasion,had induced cell apoptosis and blocked the cell cycle,which had the value of further research.2.The study of mechanism indicated that combination of afatinib and enzalutamide had a significant synergistic inhibitory effect on protein expression level of tyrosine kinase HER2,HER3 and androgen receptor AR and PSA,meanwhile the combination treatment also reverse the increase in HER2 expression and Akt phosphorylation induced by enzalutamide,which had the great anti-tumor activity in vitro.